Genetic correction of cystic fibrosis (CF) requires the development of safe and efficient vector systems capable of achieving direct in vivo gene delivery to the airway epithelium. An ideal vector for gene therapy of CF lung disease should possess the following features: 1) tissue tropism for respiratory epithelium; 2) ability to transfer genes efficiently into terminally differentiated cells; and 3) stable chromosomal integration for prolonged gene expression. Currently, a number of viral systems are available for somatic gene transfer, including the well-characterized retrovirus, adenovirus, and the relatively new adeno-associated virus (AAV). In addition, liposomes and molecular conjugates are also being explored as alternative gene delivery systems. Unfortunately, none of these current vectors possess all the features desired of an ideal gene transfer system. Two general strategies to develop clinically useful vectors for CF are proposed. First, we shall improve current transient (adenoviral) and stable (AAV) expression vectors. In Specific Aim 1, we shall improve the safety of recombinant adenovirus vectors, focussing on strategies to minimize vector replication/reactivation and to develop techniques (hexon marking) to monitor these events. In Specific Aim 2, we shall characterize AAV as a vector for CFTR delivery in immortalized and primary cultures of airway epithelial cells, focussing on integrative capacities (targeted versus random), promoters, and stability of expression. Second, our experience with the AAV- and molecular conjugate- mediated gene transfer systems indicates that a strategy based on the combined advantages of these vector systems may lead to novel approaches for CF gene therapy. In Specific Aim 3, we shall develop strategies and reagents to incorporate an integration mechanism derived from AAV into adenovirus-coupled molecular conjugates. Both technically and conceptually, these Specific Aims are closely related, providing us with an efficient, interactive approach for developing a clinically useful vector for CF gene therapy. Further, the reagents derived from this proposed study may be applicable to other inherited diseases.